G protein linked receptors (GPCRs) mediate most of the cellular actions of neurotransmitters and hormones in the body as well as the therapeutic effects of almost a third of the drugs sold by the pharmaceutical industry. GPCRs desensitize and internalize when they are continuously stimulated with agonist. This receptor signaling turn off is a major cause of tolerance development to therapeutic agents. Drugs that could prevent desensitization and internalization could prolong the therapeutic actions of many drugs in use today especially drugs used in the treatment of pain. Presently, there is no in vitro assay that measures GPCR desensitization in a manner that is amenable for drug discovery. We propose to develop a simple, sensitive assay that measures critical molecular events involved in GPCR desensitization that could be employed to discover drugs that prevent tolerance development. Specifically, we will use our highly sensitive complementation technology to detect the translocation of Beta-adrenergic receptor kinase (BARK) and B-arrestin from the cytosol to the receptor. These translocation events are critical for the initiation of the cellular cascade responsible for GPCR desensitization and drugs that block this translocation can prevent receptor desensitization. The assay will involve the use of a highly sensitive intracellular B-galactosidase (B-gal) complementation assay in which one fragment of B-gal, which by itself is inactive, is engineered into either BARK or B-arrestin and another inactive component of B-gal is targeted to the cell membrane using the CAAX farnesylation motif. When translocation of BARK or B-arrestin from the cytosol to the membrane occurs, the two components of B-gal combine intracellularly to form an active B-gal enzyme which catalyzes the synthesis of 1000s of molecules of a fluorescent product that can be visualized We will employ the same technology to measure internalization of GPCRs following long-term agonist treatment. Both assays will be optimized for high throughput screening and employed to identifying compounds that block receptor desensitization. Furthermore, these assays could have a broader role in drug discovery by identifying agonists at the large population of orphan GPCRs since stimulation of all GPCRs causes BARK and B-arrestin translocation.